Determination of Circulating Tumor Cells in Peripheral Blood By Flow Cytometry

Lunx Is a Superior Molecular Marker for Detection of Non-Small Lung Cell Cancer in Peripheral Blood

Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase Chain Reaction Method

DETECTION OF β-HUMAN CHORIONIC GONADOTROPIN EXPRESSING CELLS BY NESTED REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN THE PERIPHERAL BLOOD STEM CELLS OF PATIENTS WITH ADVANCED GERM CELL TUMOR

334P - Clinical Significance and the Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Breast Cancer

Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch (®) technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch (®) technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch (®) technology.

Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch® technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch® technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch® technology.

In 50-60 per cent of patients who undergo hepatic resection for metastasis of colorectal cancer the first site of tumour recurrence is extrahepatic, indicating the presence of more extensive disease at the time of resection. The aim of this study was to evaluate whether the presence of disseminated tumour cells in blood and bone marrow could predict extrahepatic tumour recurrence. Cytokeratin 20 (CK20) reverse transcriptase-polymerase chain reaction was used to study the presence of tumour cells in preoperative peripheral blood and bone marrow samples from 41 patients with liver metastasis scheduled for surgical resection. CK20 expression was detected in six of 41 peripheral blood samples and in eight of 32 bone marrow samples. There was no correlation between CK20-positive samples and subsequent extrahepatic recurrence. Positive blood samples did, however, correlate with high serum carcinoembryonic antigen level and large tumour volume. None of the 14 patients previously treated with chemotherapy had CK20-positive samples, whereas six of 27 blood and eight of 20 bone marrow samples were positive in the chemotherapy-naive group. Although the number of patients in this study is limited, the presence of disseminated tumour cells did not predict subsequent extrahepatic recurrence. The results strongly suggest that the presence of circulating tumour cells in peripheral blood may reflect transient shedding of tumour cells related to large tumour volume.

Quantitative RT-PCR Detection of Colorectal Tumor Cells in Peripheral Blood—A Systematic Review

The prognostic relevance of disseminated tumor cells in bone marrow of breast cancer patients at the time of primary diagnosis and during recurrence-free follow-up has been confirmed by large pooled analyses. Furthermore, circulating tumor cells (CTC) in peripheral blood have been shown as predictor of shortened progression-free and overall survival in metastatic disease. In view of the lack of data in early breast cancer, we evaluated whether the presence of CTC before the start of systemic adjuvant treatment increases the risk of subsequent relapse and death. Patients and Methods The SUCCESS A-Study is an open-label randomized controlled, phase III study comparing the disease-free survival after randomisation in patients treated with 3 cycles of Epirubicin (100 mg/m2)-Fluorouracil(500)- Cyclophosphamide (500, FEC)-chemotherapy, followed by 3 cycles of Docetaxel (100 mg/mg2, D) versus 3 cycles of FEC, followed by 3 cycles of Gemcitabine (1,000mg/m2 d1,8)-Docetaxel (75 mg/m2)(DG). In 2026 patients CTC were analyzed using the CellSearchSystem (Veridex, USA). 23 ml of peripheral blood were drawn after R0-resection of the primary tumor but before the start of adjuvant systemic treatment. After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labelled with anti-Ck8/18/19 and anti-CD45 antibodies to distinguish between epithelial cells and leukocytes. Patients were followed for a median of 35 months. Univariate and multivariable proportional hazards models were estimated to assess the prognostic significance of CTC for disease-free and overall survival. Patients with evidence of at least 1 CTC were counted as positive. Results In 21.5% of patients (n = 435) CTC were detected before the start of systemic treatment (median 1.3, range 1 - 827). Patients with CTC before treatment were more frequently node-positive (P<0.001), but no correlation to tumor size, grading and HR-Status could be found. 114 recurrences occurred and 66 patients died of their disease. The presence of CTC before systemic treatment predicted poor disease-free (p < 0.0001), distant disease-free survival (p < 0.001) and overall survival (p = 0.0002). In multivariate analysis, detection of CTC before treatment was confirmed as independent predictor for both disease-free (HR 1.88) and overall survival (HR 1.91) next to tumor size, grading, lymph node involvement and hormone receptor status (p for all < 0.05). Outcome of patients was correlated to the number of CTC. Prognosis was worst in patients with 5 CTC or more with a four-fold increased risk for recurrence and and a three-fold increased risk for death (HR 4.04 for DFS and 3.05 for OAS; p < 0.05). Conclusions This is the first study to prospectively demonstrate the prognostic relevance of CTC in peripheral blood of early breast cancer patients before the start of systemic treatment in a large patient cohort. CTC detection could serve as clinically useful prognostic marker and treatment monitoring tool and should be tested as indicator for secondary adjuvant treatment interventions within clinical trials. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-5.

This study aimed to characterize the distribution of circulating tumor cells (CTCs) in preoperative peripheral blood and intraoperative surgical lavage fluid from patients with colorectal cancer (CRC), and to evaluate the association between CTC levels and clinical prognosis to determine their potential as novel prognostic biomarkers. Preoperative peripheral blood (7.5 ml) was collected from 185 CRC patients and from 50 healthy controls. During laparoscopic surgery, 150 ml of surgical lavage fluid was obtained from each patient at three key time points: before tumor resection, after tumor resection and after mesenteric incision of the tumor. CTCs were detected using the CanPatrolTM enrichment system combined with RNA in situ hybridization. Associations between CTC counts and clinical indicators were analyzed, and their relationship with progression-free survival (PFS) and overall survival (OS) was evaluated. The CTC-positive rate in both peripheral blood and surgical lavage fluid was associated with tumor invasion depth, TNM stage and metastasis. TNM stage and CTC counts in peripheral blood and lavage fluid were identified as independent predictors of mortality. Among patients with CTC-positive lavage fluid after tumor mesangial dissection, both PFS and OS were significantly shorter compared with CTC-negative patients (PPFS=0.0015, POS=0.0013). These findings indicate that CTC counts in peripheral blood and surgical lavage fluid serve as important biomarkers for predicting CRC prognosis and are closely associated with both PFS and OS.

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2 positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

Circulating tumor cells (CTCs) in peripheral blood is an indication of poor prognosis for patients with different cancer types. However, most of the available technologies for detecting CTCs show low sensitivity and specificity. Therefore, we attempted to find an alternative marker for CTCs of colorectal cancer. We have directly extracted RNA from CTCs contained in 1.5 mL peripheral blood from 90 colorectal cancer patients and 151 healthy donors, and screened these samples for candidate marker genes by nested real-time quantitative polymerase chain reaction (PCR). From genes selected from a public database of microarray analyses, we successfully identified epithelial cell transforming sequence 2 oncogene (ECT2) as a gene that exhibits high differential expression ratios (p < 0.01). ECT2 displays good sensitivity and specificity, with an area under the curve (AUC) value of 0.821. This marker gene also has a high detection rate in patients with serum carcinoembryonic antigen (CEA) concentrations below the diagnostic threshold of 5 ng/mL. The expression of ECT2 can therefore serve as an alternative measurement that can compensate for the inadequacy of the current CEA test in the diagnosis and monitoring of colorectal cancer patients.

This study investigates the correlation between circulating tumor cells (CTCs) in peripheral blood and the clinical characteristics and prognosis of advanced pediatric neuroblastoma (NB). We conducted a retrospective analysis of 144 children with advanced NB who underwent comprehensive treatment. Detailed clinical data were collected, and CTCs were detected using a negative enrichment method combined with immunofluorescence technology. Prognostic evaluation criteria and cutoff values for CTCs were established using ROC curve analysis. Univariate and Cox multivariate regression analyses identified independent risk factors impacting prognosis. Patients were categorized into high and low-expression groups based on optimal cutoff values determined with X-tile software. The high expression group had a significantly higher incidence of disease progression (p < 0.001), maximum tumor diameter ≥10 cm (p = 0.004), undifferentiated subtype (p = 0.034), and stage IV disease (p = 0.007) compared to the low expression group. CTCs were notably higher in patients with progression compared to those with mitigation (p < 0.001), in those with maximum tumor diameter ≥10 cm compared to <10 cm (p < 0.001), and in stage IV compared to stage III patients (p = 0.036). The AUC values for maximum tumor diameter, degree of differentiation, and tumor stage were 0.703, 0.669, 0.574, and 0.598, respectively. The detection of CTCs provides significant insights into the clinical characteristics and prognosis of advanced pediatric NB, highlighting its potential as a prognostic tool.

Objective: To investigate the value of the number of circulating tumor cells (CTC) in peripheral blood in the prognosis and coagulation-related indicators of patients with renal cancer. Methods: 65 patients with renal cell carcinoma (RCC) confirmed pathologically were divided into CTC positive group and CTC negative group according to the CTC count (5 pcs/3.5 ml). Compare the age, gender, tumor location, TNM (clinical stage), pathological grade, tissue type, lymph node metastasis, distant metastasis, prognosis and prothrombin time (PT), fibrinogen (FIB), partial coagulation of the two groups of patients The correlation between the results of zymogen time (APTT) and D-dimer (DD) and the number of CTC. Results: There were significant differences in TNM, lymph node metastasis, and distant metastasis between the two groups (P < 0.05). The number of CTC in patients was correlated with FIB and D-D levels (P < 0.05). Conclusion: The number of CTC in patients with renal cell carcinoma is correlated with some clinical phenotypes (TNM, lymph node metastasis, distant metastasis) and some coagulation indexes (FIB, D-D), and can jointly predict the prognosis of renal cancer.

Homoporous polydimethylsiloxane membrane microfilter for ultrafast label-free isolation and recognition of circulating tumor cells in peripheral blood